The Repeat Expansion Diseases are caused by the intergenerational expansion of a tandem repeat at a single genetic locus. Expansion has different consequences depending on the gene involved, the location of the repeats within the gene, and the sequence of the repeat unit. Expansion of a CGG:CCG-repeat in the 5' UTR of the FMR1 gene is associated with 3 quite different clinical presentations: Individuals with >200 repeats have Fragile X mental retardation syndrome. Individuals with 60-200 repeats are at risk for 2 quite different problems, namely Fragile X-associated tremor-ataxia syndrome and premature ovarian failure. GAA:TTC-repeat expansion in the first intron of the frataxin gene causes a deficit in frataxin mRNA. This results in Friedreich ataxia, a degenerative disease associated with cerebellar dysfunction, hypertrophic cardiomyopathy, and diabetes. We are interested in both the mechanism of expansion and the consequences of expansion in these disorders. Mouse models of repeat expansion: We have previously shown that CGG:CCG-repeats are not intrinsically prone to expansion in transgenic mice. This suggests that sequence context or differences in trans-acting factors might play a role in expansion in humans. However, despite the fact that mice and humans are syntenic in the region of the X chromosome containing the FMR1 gene, we find no evidence of instability in "knock-in" mice we have generated that contain a repeat length that has a ~100% likelihood of expansion in humans. We are now examining the effect of candidate trans-acting factors for effects on instability. To date we have ruled out an effect of folate deprivation, and maternal imprinting. We have also tested the effect of defects in DNA damage surveillance genes as well as helicases that had been thought to be important in expansion. Mutations in the yeast homolog of the FEN-1 gene have been shown to cause a dramatic increase in expansion in this model organism. However, we have shown that haploinsufficiency of FEN-1 does not lead to expansion in mice. Since FEN-1 homozygous mice die very early in embryonic development, we now plan to examine repeat instability in FEN-1 null blastocysts. The molecular basis of CGG-repeat induced disease pathology: We have shown that cell lines with long CGG-repeats have elevated levels of apoptosis. Apoptosis is increased in the presence of doxycycline which induces transcription of these repeats in this cell line. This suggests that expression of RNA with a long CGG-repeat tract has deleterious consequences for cells. It is possible that this "toxicity" contributes to the cerebellar and ovarian degeneration seen in individuals with 60-200 repeats. It would also explain why such symptoms are not seen in people with >200 repeats, since it is known that in such instances the FMR1 promoter is silenced. We have gone on to show that CGG-RNA readily forms very stable hairpins containing a mixture of C:G and G:G base pairs. Such hairpins may explain the stalling of the 40S ribosomal subunit that is seen on long FMR1 alleles. These hairpins may also contribute to apoptosis caused by this RNA since RNA with double-stranded character can be toxic in a number of ways. For example, the interferon-inducible protein kinase, PKR is an enzyme that can be activated by certain double-stranded RNAs (dsRNAs). This activation leads in turn to apoptosis. We have shown that, unlike CUG-RNA hairpins formed by the repeats responsible for myotonic dystrophy type 1, the CGG-hairpins do not activate PKR. However, we have shown that these hairpins are a substrate for dicer. Dicer is part of the RNA interference (RNAi) pathway. It is the enzyme that digests certain long double-stranded RNAs to generate the small interfering RNAs (siRNAs) that are involved in the targeted destruction of homologous transcripts. Since a number of mRNAs in humans are known that contain CGG-repeats, it is possible that the toxicity of the CGG-RNA is exerted, at least in part, via the RNAi-mediated targeted degradation of such transcripts. The molecular basis of GAA:TTC-repeat-mediated frataxin mRNA deficiency in FRDA: In an effort to understand the molecular basis of the deficiency of the frataxin transcript in individuals with FRDA we have analyzed the frataxin promoter, first intron and 3' UTR in some detail. In doing so we have identified key regions that are important for normal transcription including 2 novel transcription factor binding sites. We had previously shown that the GAA:TTC repeats were able to form a triple-stranded DNA structure that impeded transcription. We now have evidence for an additional effect of these repeats on transcription that may have important implications for disease etiology.